Gatifloxacin was obtained from LKT Laboratories, Inc. (St. Paul, MN). For the cell proliferation assay, gatifloxacin was solubilized in Dulbecco’s modified essential medium (DMEM, Gibco, Grand Island, NY), which was then used as part of the culture medium described below. To evaluate minimum inhibitory concentration (MIC), the gatifloxacin was solubilized in the broth described below for liquid culture of bacteria.

For plate culture, the bacteria tested in this study were grown on plates containing tryptic soy agar (40 g・L⁻¹; Becton, Dickinson and Company, Franklin Lakes, NJ) supplemented with 10% defibrinated horse blood (Nippon Bio-Test Laboratories Inc., Tokyo, Japan), hemin (5 g・L⁻¹; Sigma-Aldrich Corp., St. Louis, MO), and menadione (0.5 g・L⁻¹; Sigma-Aldrich Corp.) [18] . Preculture was performed in an anaerobic chamber (N₂: 80%, H₂: 10%, CO₂: 10%) at 37˚C. For liquid culture, P. gingivalis ATCC 33277 and P. intermedia ATCC 25611, which were obtained from the American Type Culture Collection (Manassas, VA), were cultured in trypticase soy broth (30 g・L⁻¹; Becton, Dickinson and Company) supplemented with hemin (5 g・L⁻¹; Sigma-Aldrich Corp.) and menadione (0.5 g・L⁻¹; Sigma-Aldrich Corp.) in the anaerobic chamber at 37˚C. A. actinomycetemcomitans 310a (kindly provided by Dr. H. Ohta, Ibaraki University) was cultured in Todd-Hewitt broth (30 g・L⁻¹; Becton, Dickinson Company) supplemented with yeast extract (10 g・L⁻¹; Becton, Dickinson and Company) in the anaerobic chamber at 37˚C [19] . Each precultured colony was inoculated into a liquid broth and incubated for 2 to 4 days under the conditions described above.

Broths containing gatifloxacin were used to determine MIC. Six serial concentrations of gatifloxacin were predetermined, ranging from 0 M to 25 nM for cultures of A. actinomycetemcomitans and P. gingivalis. Those of P. intermedia were predetermined from 0 M to 1.0 × 10³ nM. Each bacterial strain was inoculated into the broth and then incubated for 3 - 7 days in an anaerobic chamber at 37˚C. Minimum inhibitory concentration was defined as the lowest concentration of gatifloxacin that would inhibit visible growth of the microorganisms after incubation. To confirm the reliability of the data, the experiments were performed four times.

Periodontopathic bacteria were anaerobically grown at 37˚C to the early stationary phase in the broth described above.

The harvested cells were washed once in aseptic broth and then resuspended in freshly aseptic broth containing sufficient concentrations of gatifloxacin. Bacterial cell suspensions were incubated at 37˚C and examined for bacterial cell viability after 6, 24, and 48 h.

Bacterial cell viability was determined by adenosine triphosphate bioluminescence assay using the BacTiter- Glo Microbial Cell Viability Assay kit (Promega, Madison, USA). Specifically, a volume of BacTiter-Glo reagent equal to the volume of each bacterial suspension was added and briefly mixed. The luminescence of the solution was then recorded by using the Gene Light Model GL-210A luminometer (Microtec Co., Ltd., Funabashi, Japan). The value obtained was expressed as the ratio to that at the start of incubation. The results were expressed as the mean ± SD of three experiments.

Normal human dermal fibroblast (NHDF) adult donor cells derived from connective tissue (Takara Bio Inc., Shiga, Japan) were used in this study. The culture medium consisted of MEM α (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS French origin; Biowest, Nuaillé, France) and antibiotics. Cells subcultured at 37˚C in a humidified atmosphere with 5% CO₂ were suspended in MEM α at a concentration of 2.5 × 10⁷ cells・L⁻¹. An aliquot of 1 × 10⁻⁴ L of cells was placed in the wells of a 96-well plate and then incubated for 4 h at 37˚C in a humidified atmosphere with 5% CO₂. The medium was replaced with medium containing gatifloxacin at concentrations adjusted by stepwise dilution. The culture medium was refreshed every two days. After completion of cultivation, cell viability was determined by using a Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) every 24 h for 4 days. Briefly, a 10-μL aliquot of CCK-8 solution was added to each well. The cells were then incubated for 3 to 4 h. Absorbance was measured at 450 nm by using a SpectraMax M5e Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA). The experiments were performed in triplicate. The data were expressed as the survival rate.

Statistical analyses were conducted by using computer software (Prism, version 5.0c; GraphPad Software, San Diego, CA, USA) for comparison across the experimental conditions. Statistical comparisons were performed using an unpaired t-test or Kruskal-Wallis test followed by Dunn’s post-hoc test.

As shown in Table 1, gatifloxacin showed an inhibitory effect on the growth of all the periodontopathic bacteria tested, with the inhibitory effect greatest on the growth of the A. actinomycetemcomitans strain (Table 1(a)). The MIC value for A. actinomycetemcomitans was 2.5 nM, and that for P. gingivalis was 25 nM, while that for P. intermedia required a higher concentration at 7.5 × 10² nM (Table 1(b)). These findings suggest that gatifloxacin exhibits selective inhibitory action against the growth of periodontopathic bacteria. To further investigate its potential to inhibit the growth of periodontopathic bacteria, the bactericidal activity of gatifloxacin was assessed. As shown in Figure 1, gatifloxacin showed bactericidal activity in a time-dependent manner against all bacteria tested. A highly significant difference was observed in the inhibitory action between cultivation time

and drug concentration using the unpaired t-test against all bacteria tested. The number of viable bacterial cells decreased significantly as the bacteria was exposed to gatifloxacin (p < 0.01) during the time tested. At 1.0 × 10³ nM, higher than the MIC, gatifloxacin reduced the number of A. actinomycetemcomitans cells after 48 hours to less than 3% of that at the start of cultivation. (Figure 1(a)). It also inhibited the growth of an even larger number

of P. intermedia (to less than 2%) and P. gingivalis cells (to less than 1%) (Figure 1(b) and Figure 1(c)). These data support the possibility that exposure to gatifloxacin suppresses bacterial growth over time, indicating that the drug may exert an inhibitory effect on the growth of periodontopathic bacteria.

As shown in Figure 2(a), gatifloxacin had no effect on cell proliferation of NHDF cells in concentrations of 1.0 × 10³ nM or less after 2-d culture. Almost all seeded NHDF cells survived after 2-d culture (Figure 2(a)), and even 81% of the cells survived after 4-d culture (Figure 2(b)), at 1.0 × 10³ nM, which concentration was higher than the MIC of bacteria tested. A highly significant difference was observed in the survival rate between 1.0 × 10⁴ nM and control (p < 0.05), and between 1.0 × 10⁵ nM and control (p < 0.01).

The results of antibacterial activity and cell viability assays suggested that gatifloxacin had bactericidal effects on A. actinomycetemcomitans and P. gingivalis at low concentrations, while it did not damage mammalian cells. The drug also exhibited anti-bacterial effects on P. intermedia at 7.5 × 10² nM, while approximately 100% of mammalian cells survived after 2-d incubation, as well as 82% after 4-d incubation. This suggested that this drug would be harmless at an MIC of 7.5 × 10² nM, also without exception in the case of P. intermedia in mammalia, especially under conditions of short-term local administration. In this study, we found that gatifloxacin had an inhibitory effect on periodontal pathogens. Further investigation was necessary, however, to elucidate the properties of this local administration drug, using the sustained release complexes established at our laboratory [20] . Moreover, the immobilization methods employed on surfaces of dental materials must be examined with reference to this drug. Of interest, also, are the functional mechanisms involved in its inhibition of the growth of periodontopathic bacteria. One of our goals is to develop a defense system against peri-implantitis. We believe that the application of gatifloxacin onto dental implants will offer advantages in the prevention of periodontal diseases such as peri-implantitis, thereby improving oral care.